Identification of a putative membrane‐interacting domain of CTP: Phosphocholine cytidylyltransferase from rat liver

FEBS Letters

Orfanos, Constantin E.²  Becker, Andreas¹  Geilen, Christoph C.²  Wieder, Thomas²  Wieprecht, Marcus³ 

[1]Institute of Cellular and Molecular Biology, Schering AG, Berlin, Germany

[2]Department of Dermatology, University Medical Center Steglitz, Free University of Berlin, Hindenburgdamm 30 12200 Berlin, Germany

[3]Department of Molecular Biology and Biochemistry, Free University of Berlin, Arnimallee 22, 14195 Berlin, Germany

关键词: CTP: phosphocholine cytidylyltransferase;    Membrane binding;    Peptide-specific antibody;    BCA;    bicinchoninic acid;    BSA;    bovine serum albumin;    CT;    CTP:phosphocholine cytidylyltransferase;    ECL;    enhanced chemiluminescence;    ELISA;    enzyme-linked immunosorbent assay;    PBS;    phosphate-buffered saline;    PC;    phosphatidylcholine;    SA2;    polyclonal peptide-specific antibody against cytidylyltransferase residues 1–17;    SA209;    polyclonal peptide-specific antibody against cytidylyltransferase residues 247–257;    S.D.;    standard deviation;   

DOI  :  10.1016/0014-5793(94)00433-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A putative membrane-interacting domain of CTP:phosphocholine cytidylyltransferase (CT) was identified using two peptide-specific antibodies. One antibody (SA2) was raised against the N-terminus of CT (amino acid residues 1–17) and the other antibody (SA209) against an α-helical domain of the enzyme (amino acid residues 247–257). Both antibodies quantitatively immunoprecipitated CT from rat liver cytosol and showed specificity towards CT when octylglucoside extracts of rat liver cytosol were assessed by Western blot analysis. However, further experiments revealed that the antibodies had different characteristics. Whereas the antibody directed against the N-terminus of CT (SA2) did not influence CT/membrane interaction, the new antibody (SA209) against the α-helical domain of the enzyme interfered with this interaction. Our results provide experimental evidence that the α-helical domain (amino acid residues 228–287) of CT may serve as a membrane-interacting domain.

【 授权许可】

Unknown